AIM: To analyze concentrations of vascular endothelial growth factor (VEGF) and fibrosis-related factors in vitreous fluid of proliferative diabetic retinopathy (PDR) patients pre-treated with intravitreal anti-VEGF injections (IVI) at different time periods prior to pars plana vitrectomy (PPV), and their correlation with the degree of vitreoretinal fibrosis and explore the optimal timing of preoperative IVI. METHODS: The prospective case-control study from January 2019 to July 2020 included 31 eyes with PDR-related complications (PDR group) and 21 eyes with non-diabetic ocular disease (control group) requiring PPV. PDR eyes were divided into four groups based on timing of PPV: 3d after IVI (3-day group); 5d after IVI (5-day group); 7 or more days after IVI (≥7-day group); and no IVI. Vitreous fluid samples (0.5-1.0 mL) were collected prior to switching on the infusion before routine 23-G PPV. Concentrations of VEGF, basic fibroblast growth factor (bFGF), periostin (PN), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were measured by immunoassay, and concentration differences for each cytokine were compared among the groups. The degree of vitreoretinal fibrosis was graded intraoperatively, and the correlation between the changes in cytokine levels and the severity of vitreoretinal fibrosis was analyzed by univariate ordinal logistic regression analysis. RESULTS: PDR eyes without IVI had significantly higher VEGF, bFGF, PN, and IL-6 concentrations than non-diabetic eyes (all P<0.05), and had a significantly higher concentration of VEGF (P<0.05) and a significantly lower concentration of IL-8 (P<0.05) than PDR eyes with IVI. Statistically significant differences were also observed for concentrations of VEGF, bFGF, PN, IL-6, and IL-8 among 3-day, 5-day, and ≥7-day groups (all P<0.05); meanwhile there was no significant difference in TNF-α among groups (P=0.226). The 5-day group had the lowest concentration of VEGF and the ≥7-day group had the highest concentration of bFGF and PN. The degree of vitreoretinal fibrosis was significantly higher in the ≥7-day group compared to the 3-day (P=0.015) and 5-day group (P=0.039), and vitreoretinal fibrosis correlated significantly with concentrations of bFGF, PN, IL-6, and IL-8 (all P<0.05). Univariate ordinal logistic regression analysis showed that bFGF was an independent risk factor for the severity of vitreoretinal fibrosis in PDR patients pre-treated with IVI. CONCLUSION: The vitreous concentrations of VEGF, bFGF, PN, IL-6, and IL-8 change after pretreatment with IVI before PPV in PDR patients. The degree of vitreoretinal fibrosis is higher in patients with a longer time between IVI treatment and PPV, which may be related to the angio-fibrosis switch. The results suggest that PPV should be performed 5d after IVI administration in PDR patients.